Glycosyl hydrolases (GHs) are a large group of enzymes that cleave glycosidic bonds between individual carbohydrate monomers in large polysaccharide molecules. For example, cellulases cleave the beta 1-4 bond between glucose monomers in the cellulose polymer; arabinofuranosidases cleave the alpha 1-2 and/or alpha 1-3 bonds between arabinose and xylose in arabinoxylan; amylases cleave the alpha 1-4 bonds between glucose molecules in starch, etc. As a result of the diversity of polysaccharide molecules, there are also many different GH enzymes. However, these enzymes all share one of two common mechanisms, called inverting and retaining, for introducing a water molecule at a glycosidic bond thus cleaving the polysaccharide. The majority of GH enzymes utilize the retaining mechanism.
The GH enzymes are grouped into more than 100 different families based on commonality in their primary and tertiary structures and their catalytic mechanism (CAZy website, URL: cazy.org: Coutinho and Henrissat, 1999). Some GH enzymes families are grouped into larger clans. Depending upon the particular family (all numbers are according to the CAZy website as of 13 March 2008), it may have only a few known examples (e.g., family GH82) or many (e.g. family GH34); more than half of the families have fewer than 200 members. Similarly, all the members of a particular family may represent essentially a single activity, which is to say activity against a single specific substrate (e.g., GH11, all of which are xylanases), whereas other families may have enzymes that cover a wide range of activities (e.g., GH5, comprising cellulases, xylanases, mannanases, chitosanases, galactanases, etc.). Individually, most enzymes have their highest activity for a single substrate, although there are examples of particular enzymes that have high activity against several substrates (e.g. Cel7B from Trichoderma reesei, which has both cellulase and xylanase activity).
The GH Family 6 belongs to no clan; it comprises over 100 members, all of which exhibit primarily cellulase activity using the inverting mechanism. Both endo- and exo-cellulases have been identified from a variety of bacterial and fungal sources. In addition, some GH6 members, including Cel6A from Trichoderma reesei, have been shown to have hydrolytic activity against beta-glucan, which is a linear polymer of glucose with mixed linkages (Henriksson et al., 1995).
The beta-glucans form a large group of industrially important polysaccharides. Because of their mixed linkages, the beta-glucans have higher solubility in aqueous solutions than more regular polymers such as cellulose. In the soluble form, the beta-glucans confer viscosity and/or gel-like properties to a solution. There are two major types: beta 1-3, 1-6 glucan, also known as laminaran because a major source of this glucan is Laminaria brown algae (kelp), and beta 1-3, 1-4 glucan, also known as lichenan because a major source of this glucan is lichen. However, beta 1-3, 1-4 glucan is also found as a major component of oat and barley endosperm. Hydrolysis of beta 1-3, 1-4 glucan from grains is desirable on the industrial scale to reduce viscosity in processes such as brewing, in the production of grain ethanol for fuel, and also to increase nutrient accessibility in animal feeds. In particular, Trichoderma reesei Cel6A expressed in brewer's yeast is used to aid in the malting and brewing processes (Enari et al., 1987).
Some efforts to engineer GH enzymes in order to switch their activity from one substrate to another have been made, although experts in protein engineering generally concede that this is one of the more difficult protein engineering challenges (cf. Tao and Cornish, 2002). The research group of W. M de Vos identified three key amino acid residues of a GH1 beta-glucosidase that determined substrate specificity based on a structural comparison to a beta-galactosidase from the same family. By converting the residues of the beta-glucosidase to those found in the beta-galactosidase, they converted the beta -glucosidase into a beta-galactosidase. Similarly, key residues of a GH10 xylanase that discriminate between xylan and cellulose have been identified and mutagenized to change the enzyme from a xylanase to a cellulase (Andrews et al., 2000).
The GH6 family of enzymes have been the target of mutational and protein engineering studies. The exocellulase Cel6A from Trichoderma reesei, the exocellulase Cel6A from Humicola insolens, and the cellulases Cel6A (endo) and Cel6B (exo) from Thermobifida fusca are representative enzymes that have been particularly well characterized. Specific sites of investigation include what are known as the loop regions. These are the principal determinants of whether an enzyme is an endocellulase (lacking loops) or an exocellulase (possessing loops). Mutations in the loops (Varrot et al., 2002) or deletion of the loops (Meinke et al., 1995) will alter the interaction between Cel6A and cellulose. An extensive series of point mutations were studied in the two T. fusca enzymes, Cel6A and Cel6B (Zhang et al., 2000a; Zhang et al., 2000b). Changes in the relative activities towards different substrates—specifically filter paper, carboxymethyl cellulose, swollen cellulose and bacterial microcrystalline cellulose—were observed. Other studies have examined the role of aromatic amino acids in substrate binding (Koivula et al., 1996; Koivula et al., 1998; Zou et al., 1999) and the role of charged amino acids in activity and stability (Koivula et al., 2002; Wohlfahrt et al., 2003).
T. reesei Cel6A (or TrCel6A) is one of the two major cellobiohydrolases secreted by this fungus and has been shown to be efficient in the enzymatic hydrolysis of crystalline cellulose, with low but measurable activity in the hydrolysis of beta 1,3-1,4 mixed linkage glucans such beta-glucan and lichenan. The tryptophan amino acid residue at position 367 (W367) of the Trichoderma reesei Cel6A represents a highly conserved residue within a strongly conserved region of the enzyme (FIG. 1). Generally, mutation of conserved residues results in enzyme inactivation, or a severe loss of activity.